The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid beta protein (Abeta), a small peptide derived from beta- and gamma-secretase cleavages of the amyloid precursor protein (APR). The production of Abeta requires APR endocytosis and subsequent trafficking to intracellular compartments where the proteolytic enzymatic activities reside. Recently, it was demonstrated that both beta- and gamma-secretase activities of BACE1 and presenilins, respectively, are concentrated in cholesterol-rich lipid raft microdomains wherein Abeta is generated. However, the mechanism by which APP is sorted to lipid rafts is unknown. Increasing evidence suggests that the Low-density lipoprotein receptor- related protein (LRP) facilitates amyloidogenic processing of APP, although the precise mechanism underlying this activity is still unclear. We recently demonstrated that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether is sufficient to elevate Abeta production by increasing BACE cleaved APP beta-CTF. This led us to hypothesize that LRP-ST might function to facilitate the intracellular sorting of APP and/or BACE into lipid raft membranes. Indeed, our preliminary results have confirmed this very prediction and provided strong evidence the endogenous LRP also normally functions to faciliate APP sorting to lipid rafts. We hypothesize that LRP within the cytoplasmic tail contains a lipid raft-targeting domain separate from the APP binding domain that enhances sorting of APP to lipid rafts. In this proposal, we aim to dissect the sequences in LRP-ST required for its physical association with lipid raft membranes and utilize this information to direct APP sorting away from lipid rafts and screen for candidate proteins responsible for lipid raft localization of LRP. The cellular and biochemical mechanisms by which Abeta is generated is critical for designing therapeutic strategies for AD. One potential therapeutic design might be to inhibit the sorting of APP to lipid raft microdomains where Abeta is generated. Based on our results, LRP is the first pathway in which the trafficking of APP to lipid rafts is affected. Thus, understanding the nature of LRP/lipid raft interaction via identification of the raft targeting sequence and interacting raft targeting protein(s) might yield important information as to how APP trafficking can be therapeutically modified to inhibit Abeta generation. [unreadable] [unreadable] [unreadable]